The overarching goal of this application is to identify inhibitors of norovirus 3C-like protease (3CLpro) as effective antiviral and prophylactic agents against noroviruses, the main causative agents of acute viral gastroenteritis. Noroviruses constitute an important public health problem as well as a potential bioterrorism threat. Currently there is no specific preventive or therapeutic measure against norovirus infections. Vaccine development for noroviruses faces several challenges including viral diversity and short-term immunity; consequently, there is an urgent need for the development of effective anti-noroviral drugs. We have initiated a norovirus research program aimed at developing small molecule therapeutics for the treatment and/or prevention of norovirus infections. Toward that end, we have identified a series of transition state inhibitors of norovirus 3CLpro and have demonstrated that the viral enzyme is a validated target for the development of anti-norovirus drugs. The inhibitors were characterized for antiviral activity against noroviruses and related caliciviruses, mechanism of action, X-ray crystallography, viral resistance, preliminary absorption, distribution, metabolism and excretion properties, tolerability, and oral availability in rats. Moreover, a protease inhibitor in the series was shown to be effective in the gnotobiotic pig model of norovirus infection. These results provide strong support for our working hypothesis that norovirus 3CLpro is a validated druggable viral target for the discovery of small molecule anti-norovirus therapeutics and prophylactics. The series of compounds are suitable for further optimization; consequently, we propose to embark on an optimization campaign that encompasses basic and applied studies and optimally integrates the deployment and utilization of multiple tools and methodologies to identify preclinical candidates. The ultimate long term goal of this program is the development of antiviral therapeutics against norovirus infection by advancing a drug candidate through the stage of filing for an investigational new drug (IND) application.